Molecular Cloning Of Estrogen Receptor Genes And Their Expression Upon Endocrine Disrupting Chemicals Exposure In Rare Minnow

Endocrine disrupting chemicals (EDCs) have the role of hormone and could interferewith the normal endocrine function of organism, including human. Environmental estrogensbelong to an important part of EDCs. Estrogens play an important role in many physiologicalprocesses of vertebrates, mediated by estrogen receptors (ERs). Estrogens have interactionwith estrogen receptors and form the estrogen-ER complex, which causes the conformationalchange of the estrogen receptors, binds to the estrogen response elements of the upstreamsequence of target genes, and enhances and regulates the transcription of target genes.Thereby, it affects the level of encoding protein of target genes and regulates the growth,development and reproductive activity of vertebrates. There are three subtypes of ER gene inmost fish, ERα, ERβ1and ERβ2respectively, which are involved in sex determination anddifferentiation and other physiological processes. Rare minnow (Gobiocypris rarus) is anunique small fish of our country and belongs to the family of cyprinidae. Rare minnow hasbeen widely used as an important animal for aquatic toxicity studies and is an ideal mode fishfor the study of EDCs.In order to elucidate the effects of endocrine disrupting chemicals (EDCs) on ER genesmRNA and explore the mechanism of action of EDCs in the vertebrates, full-length cDNAsequences of ERα, ERβ1and ERβ2were isolated and characterized from the rare minnowusing homology cloning and RACE methods. The fragments of three ER transcripts werealigned to generate a2438-bp ERα, a2724-bp ERβ1and a2600-bp ERβ2, encoding a598, a608and a554amine acid polypeptide, respectively. Sequences analysis show that the threegenes are the members of nuclear receptor family and have the same functional and structuraldomains like other nuclear receptors, including six domains, A/B, C, D, E and F regions fromthe N-terminus to the C-terminus. The C domain and E domain are highly conserved and theyare DNA binding domain and ligand binding domain respectively. Multiple alignment andphylogenetic tree analysis indicated that rare minnow ERs shared the highest amino acididentities with counterparts of three cyprinidae species (Pimephales promelas ERα:91.1%,Rutilus rutilus ERβ1:92.9%, Tanichthy albonubes ERβ2:93.5%) and ER isoforms are morerelated to counterparts of cyprinidae species. The semi-quantitative RT-PCR method was used to detecte the tissue distribution of ERα, ERβ1, and ERβ2genes. The expression of ERαmRNA was high in gonad and liver. The ERβ1transcript was the highest in the liver of femalefish and was evenly high in the liver, testis and intestine in male. The ERβ2transcript washigh in liver, gonad, and intestine.The rare minnow juveniles of34days post fertilization were exposed to EDCs (EE2, NPand BPA) and each EDCs contains three concentrations, EE2(0.01,0.1,1nM), NP (10,100,1000nM) and BPA (0.1,1,10nM). After3days exposure, quantitative real-time PCR wasused to investigate the change of ERα, ERβ1and ERβ2gene mRNA expression. Compared tocontrol, EE2exposure at0.01,0.1and1nM extremely significant increase ERα transcript.ERβ1gene mRNA expression was suppressed by EE2at all concentrations and reached to thelevel of significant at1nM-EE2. However, ERβ2transcript had opposite response to EE2atlow and high concentrations (up-regulation at0.1nM and down-regulation at1nM,significantly). Except a weak increase of ERα at10nM NP, varying decrease of three ERtranscripts was resulted in by NP at10,100and1000nM. BPA as a weak estrogenic effect ofEDCs, has no obvious effects on ERα, ERβ1and ERβ2gene mRNA expression except thatERα transcript was significantly up-regulated by BPA at10nM and ERβ2transcript wassignificantly down-regulated by BPA at1nM. The BPA exposures at other concentrationsalmost had no effect on the ER transcripts.These results combined suggested that the ER genes are not modulated in the samemanner by EE2, NP, and BPA. Different ER subtypes also may be involved in other pathwaysand they are coordinate with each other to regulate the growth, development and reproductionof fish.